Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Summary

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Abstract

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Introduction

Fungal exposures in indoor and occupational environments can result in respiratory morbidities, including allergic sensitization and asthma¹. Identification of fungal hazards is important for assessing risk and preventing worker exposure. These fungal hazards may be a result of indoor contamination, outdoor air intrusion, or environmental disturbances that result in the transport of fungal materials into areas where workers are present². Methods to assess fungal exposure have included viable culture sampling as well as microscopic identification of fungal spores. These approaches have several limitations and often overlo....

Protocol

1. Preparing the NIOSH aerosol sampler

NOTE: The NIOSH aerosol sampler is a two-stage cyclone aerosol sampler that collects bioaerosols using two sampling tubes and a polytetrafluoroethylene (PTFE) filter.

1. Before assembly, carefully inspect the sampler. Check the sampler to ensure it is not damaged, all screws are in place and snug, and the sealing tape around the seam formed by the two halves is intact. Inspect the sampler O-ring to ensure it does not have any nicks, cracks or tears and that it has a very light coating of silicone grease.
   NOTE: The sampler includes a 37 mm 3 µm PTFE filter in a polystyrene or polypr....

Results

The species distribution within an environment can be assessed using relative abundance by determining the number of clones of each OTU identified in the air samples. Figure 6 is a Krona chart representing the taxonomically placed species within an indoor environment following 60 min of air sampling. It can be observed that the environment contains a variety of species within two major fungal phyla, Ascomycota and Basidiomycota, as well as species belonging t.......

Discussion

Determining the fungal diversity within an occupational environment using sequencing-based approaches has improved fungal hazard identification and exposure assessment. Using this approach has allowed for the detection of many additional fungal species that are often not detected using culture or microscopy-based methods of assessment. A method for sampling bioaerosols from occupational and indoor environments and the extraction of genomic DNA from air samples for ITS amplification and sequencing is presented here. Deter.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NIOSH BC251 bioaerosol cyclone sampler	NIOSH	BC251	The NIOSH sampler is not yet commercially available. Please contact William Lindsley, PhD (wlindsley@cdc.gov) for information on obtaining the NIOSH sampler
Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps	Fisher Scientific	02-681-373	1.5 mL polypropylene microcentrifuge tubes for air sampling; screw top threading must match the threading of the NIOSH sampler
Falcon 15 mL Conical Centrifuge Tubes	Corning	352096	15 mL polypropylene tubes for air sampling
Clean Room Vinyl Tape, Easy-Remove, 1/4" Width	McMaster-Carr	76505A1	sealing tape
Filter Cassette, Clear Styrene, 37 mm	SKC Inc.	225-3LF	3-piece sampling cassette (no filter). Contains: cassette base, extension cowl, cassette cap and inlet/outlet plugs
PTFE hydrophobic fluoropore membrane filters, 3.0 µm, 37 mm	EMD Millipore	FSLW03700	Contains: 37 mm, 3.0 µm PTFE filters and support pads
Fisherbrand filter forceps	Fisher Scientific	09-753-50	filter forceps
Model 502 Precision PanaPress	PanaVise	502	pneumatic cassette press is constructed from this precision arbor press
Scotch Super 33+ vinyl electrical tape	McMaster-Carr	76455A21	19 mm tape
Multi-purpose Calibration Jar, Large	SKC Inc.	225-112	calibration jar
Universal PCXR4 Sample Pump	SKC Inc.	224-PCXR4	sampling pump
Mass Flowmeter 4140	TSI Inc.	4140	flow meter
Roche High Pure PCR Template Kit	Roche Diagnostics	11796828001	Kit used for genomic DNA extraction. Contains: Lysis buffer, Binding buffer, Proteinase K, Inhibitor removal buffer, Wash buffer, Elution buffer, Glass fiber filter tubes and 2 ml collection tubes
Fisherbrand 2 mL Reinforced Polypropylene Screw Cap Tubes with Caps	Fisher Scientific	15340162	2 mL reinforced tubes for bead homogenization
Glass beads, acid washed, 212-300 µm	Sigma-Aldrich	G1277	glass beads
Fisher Scientific Bead Mill 24 Homogenizer	Fisher Scientific	15-340-163	bead homogenizer
CelLytic B Cell Lysis Reagent, 10X	Sigma-Aldrich	C8740	lysis reagent
Platinum Taq polymerase	Invitrogen	10966-018	Contains: Platinum Taq polymerase, 10X PCR buffer (no MgCl2), 50 mM MgCl2, KB Extender
dNTP Mix	Invitrogen	18427-088	10 mM dNTP mix
QIAquick PCR Purification Kit	Qiagen	28106	Kit used to purify fungal amplicons. Contains: Buffer PB (binding buffer), Buffer PE (washing buffer), Buffer EB (elution buffer), pH Indicator dye (optional), and GelPilot loading dye
Owl EasyCast Mini Gel Electrophoresis System	Thermo Fisher	B1 or B2
TrackIt 1 KB Plus DNA Ladder	Thermo Fisher	10488-085	DNA ladder